A number of biological activities are regulated by the interactions of hormones, polypeptide growth factors, or other agents (including light) with specific cell surface receptors. Many of these receptor-coupled signal transduction pathways are compressed of at least three types of components: the receptor protein, itself, a GTP-binding protein (G protein) which serves as a transducer in mediating receptor-regulation and an effector enzyme or an ion channel which is responsible for maintaining the level of a specific cellular second messenger. The studies outlined in this proposal will use the retinal visual transduction system as a model for addressing a number of issues relevant to receptor-G protein- effector interactions. Each of the primary components of the vision system (i.e. the photoreceptor, rhodopsin, the G protein, transducin, and the effector enzyme, the cyclic GMP phosphodiesterase (PDE)) will be purified and their functional interactions reconstituted in well defined phospholipid vesicle systems, fluorescence spectroscopic techniques, together with a variety of biochemical approaches, will be used to probe receptor- G protein and G protein-effector coupling in these systems. This proposal is divided into four specific aims: 1) the characterization of the individual steps of the receptor (rhodopsin)-stimulated activation-deactivation cycle of a G protein (transducin), 2) structure-function characterization of the phosphorylation of the alpha subunit of transducin, 3) structure- function studies of the G protein-beta gamma subunit complexes, and 4) the characterization of the molecular mechanisms by which G proteins regulate the activities of effector proteins. Among the specific issues which will be addressed include: a) the detailed sequence of events resulting in receptor-stimulated guanine nucleotide exchange, G protein-subunit dissociation, G protein- effector interactions, and the determination of the rates for these steps, using recently developed fluorescence approaches, b) whether the phosphorylation of G proteins serves as a means to regulate the function of these transducers, c) the individual roles of the beta and gamma subunits of G proteins in promoting the receptor- stimulated activation of G-alpha subunits, d) whether the receptor protein directly accentuates (via a receptor-G protein complex) G protein-effector interactions and e) the mechanism by which the GDP-bound form of the G protein-alpha subunit results in an immediate deactivation of effector activity. In addition, the reconstitution of a G protein-mediated inhibition of a verapamil- sensitive Ca2+ channel has recently been achieved and this system will be used to examine the similarities between G protein-channel interactions and transducin-PDE interactions. It is expected that the results of these studies will be relevant to understanding the molecular mechanism underlying visual transduction as well as other receptor-coupled signaling systems such as those responsible for the regulation of adenylate cyclase, phosphoinositide lipid turnover, ion channels and growth factor action.